Epidermal growth factor (EGF) is a polypeptide capable of stimulating proliferation of a wide range of cell types in vitro. In an attempt to further purify EGF from various commercial sources, we noticed that multiple forms of the polypeptide could be demonstrated by reversed-phase high-performance liquid chromatography. The two major forms, Alpha- and Beta-EGF, were analyzed to determine any properties unique to that originally reported for EGF. The physicochemical properties of the two peptides were essentially identical but analysis of the two peptides by mass spectrometry and amino acid sequencing indicated the Alpha-EGF had the primary structure originally reported for EGF, and that Beta-EGF was missing the amino-terminal asparagine. This [Des-Asn1]-EGF was significantly less potent than Alpha-EGF in stimulating proliferation of human embryonic palatal mesenchyme cells. In a second study, 125 I-Alpha-EGF was injected intravenously to examine the potential of this peptide to cross the placental barrier in CD-1 mice at different stages of pregnancy (10, 13, and 17 days). At the earliest periods that counts could be detected in embryos (5-10 min) after injection of the trace, embryos were removed, extracted, and the extracts examined by gel filtration; only free 125I was detected. Intact 125I-Alpha-EGF could not be found in plasma or other tissues 5 min after injection. In vitro studies indicate that the peptide can be rapidly degraded to amino acids plus monoiodotyrosine (MIT) by the placenta; some tissues, such as the kidney, can dehalogenate MIT to account for the free 125I that crosses the placenta. The intact peptide, however, exhibits no placental transfer. In a third study, we are exploring the potential role of EGF to mediate estrogen-induced proliferation of uterine epithelial cells. Our present data suggest that mouse uterine epithelial cells in vitro are responsive to EGF, exhibit specific binding sites, and reveal positive cytoplasmic immunolocalization of the polypeptide in vivo.